Control of chemical reactions using isotachophoresis

ABSTRACT

Isotachophoresis (ITP) is exploited to control various aspects of chemical reactions. In a first aspect, at least one of the reactants of a chemical reaction is confined to an ITP zone, but the resulting product of the chemical reaction is separated from this ITP zone by the ITP process. In a second aspect, one or more reactants of a chemical reaction are confined to an ITP zone, and one or more other reactants of the chemical reaction are not confined to this ITP zone. In a third aspect, ITP is employed to confine at least one reactant of a chemical reaction to an ITP zone, and at least one reactant of the chemical reaction is delivered to the ITP zone in two or more discrete doses. These aspects are especially relevant to performing polymerase chain reactions using chemical denaturants as opposed to thermal cycling.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 12/587,537 filed Oct.7, 2009, and hereby incorporated by reference in its entirety. U.S. Ser.No. 12/587,537 claims the benefit of U.S. provisional patent application61/195,395, filed on Oct. 7, 2008, entitled “Methods and Apparatus forControlled Chemical Cycling, Isothermal Polymerase Chain Reaction”, andhereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to control of chemical reactions.

BACKGROUND

Chemical reactions are frequently controlled in specialized ways inorder to provide various benefits, such as improved yield, increasedreaction rate, analysis of products, etc. One example of an importantreaction that is often subject to specialized control is the polymerasechain reaction (PCR). PCR is itself a multi-reaction process which mayinclude several types of chemical processes including DNA denaturation,primer annealing, primer extension (with the aid of an enzyme), and forreal time detection may include side reactions such as hybridization(e.g., with a fluorescently labeled oligonucleotide) or intercalation ofa fluorescent molecule into polynucleotide. PCR is an essential tool inboth biology and medicine, and is the technique of choice for DNAamplification. It is commonly used for the identification as well asquantification of nucleic acids or polynucleotides, in particular ofdeoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Exemplaryapplications are diagnosis of hereditary disease, forensics, geneexpression profiling and pathogen detection.

At the present time, most PCR efforts use “thermal cycling”. In thismethod, double-stranded nucleic acid is subjected to a three-stepthermal cycle where it is denatured, annealed, and extended by theaction of a thermostable DNA polymerase. In a typical thermal cyclingprocess, the reaction temperature is cycled between 55 and 94 degreesCelsius, thus reaching a point where ordinary polymerases typicallydenature. Conventional thermal PCR requires costly and complex equipmentand can be difficult to automate in miniaturized devices. It requiressignificant instrumentation, thermal control, and an expensive,thermostable DNA polymerase. Attempts have been made to avoid thermalcycling in PCR. For example, chemical denaturation (as opposed tothermal denaturation) is considered in U.S. Pat. No. 5,939,291 and in US2008/0166770.

Electrokinetic and microfluidic technology have been demonstrated forcontrolling some aspects of chemical reactions. For example, in US2008/0000774, several methods for controlling the concentration ofchemical reactants in a microfluidic system are considered. Enhancingthe concentration of a reactant is often referred to as “focusing” thereactant.

Although it would be attractive to provide for isothermal PCR in aminiaturized fluidic system, conventional fluidic approaches tend tohave difficulty with the specialized requirements of PCR (e.g., thelarge number of reaction cycles, and the need for tight control ofreactant and/or product location). Accordingly, it would be an advancein the art to provide improved chemical reaction control, especially inrelation to microfluidic PCR.

SUMMARY

In the present approach, isotachophoresis (ITP) is exploited to controlvarious aspects of chemical reactions. In ITP, a sample of one or moreanalytes is typically introduced between a leading electrolyte (LE,containing a leading ion) and a trailing electrolyte (TE, containing atrailing ion). The leading ion, trailing ion and sample components allhave the same charge polarity, (i.e., are all anions or cations).Typically, the sample components have effective electrophoretic mobilityless than that of the leading ion, but greater than that of the trailingion. Initially, the sample can be mixed with the LE, with the TE,between the LE and TE, or with both the LE and TE. On application of anelectric potential to this system, sample components migrate toward theregion between the LE and TE. Typically, these sample ions then formdiscrete contiguous zones of analyte arranged in order of their(effective) electrophoretic mobilities with the highest mobility nearestthe LE. Further details relating to isotachophoresis are described in atext “Isotachophoresis: theory, instrumentation, and applications” byauthors Everaerts, F. M., J. L. Beckers, et al., published in Amsterdamand New York by Elsevier Scientific Pub. Co. in 1976, and herebyincorporated by reference in its entirety.

The simplest applications of ITP to controlling chemical reactions areenhancing the concentration of a reactant and using ITP to move achemical reaction zone through a system. However, ITP also allows forcontrol of several other significant aspects of chemical reactions, andit is these further aspects that are of interest here.

In a first aspect, at least one of the reactants of a chemical reactionis confined to an ITP zone, but the resulting product of the chemicalreaction is separated from this ITP zone by the ITP process. Thisamounts to simultaneous reaction and separation operations, where areaction takes place in the ITP zone, and the product is separated fromthis zone. The product can be unconfined by the ITP, or it can beconfined by the ITP to a separate ITP zone than the reactant zone.

In a second aspect, one or more reactants of a chemical reaction areconfined to an ITP zone, and one or more other reactants of the chemicalreaction are not confined to this ITP zone. This amounts to a situationwhere the un-confined reactants can “flow through” the ITP zone wherethe reaction takes place. For example, a species with effective mobilityhigher than the LE can be injected in the TE reservoir. It will thenelectromigrate through the TE zone, through the sample zone(s), andfinally through the LE zone in the channel and into the LE reservoir.

In a third aspect, ITP is employed to confine at least one reactant of achemical reaction to an ITP zone, and at least one reactant of thechemical reaction is delivered to the ITP zone in two or more discretedoses.

All three of the above aspects are relevant to and offer substantialadvantages in connection with a preferred embodiment where PCR iscarried out using chemical denaturants. It is convenient to refer tosuch PCR reactions as chemically cycled PCR (ccPCR). The nucleic acidsand primers/oligonucleotides of the PCR reaction are confined by ITP,while repeated doses of a nucleic acid denaturant flow through the ITPzone. In many cases, it is preferable to control the ITP zone motion sothat it is substantially stationary relative to the walls of the liquidchannel. This can be done by opposing the sample electromigration with apressure-driven and/or electroosmosis-driven counter-flow of thesolvent. This can be accomplished by matching the ITP migration velocitywith an equal and opposite area-averaged velocity of the solvent (thebulk liquid) in the channel. Creating a substantially stationary ITPzone with respect to the lab can significantly simplify controlling thetiming and method of injecting discrete reactant doses into the channeland the monitoring of concentrations of reactants and/or products, e.g.,using fluorescent tags. Catalysts and/or enzymes can be provided in theITP reaction zone in order to alter reaction rates, typically toeffectively increase rates.

The ITP process may be able to simultaneously provide the PCR reactionand separation of PCR reaction constituents. For example, nucleic acidtemplates can be separated from oligonucleotides by confinement to twoseparate ITP zones. In some cases an electrophoretic spacer ion can beadded that forms a separate ITP zone between the nucleic acid templatezone and the oligonucleotide zone, thereby further enhancing theseparation of PCR reaction constituents. Preferably, the PCR reaction iscarried out at constant temperature, thereby avoiding the complexitiesassociated with thermal cycling.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a conceptual representation of chemical cycling PCR.

FIG. 2 illustrates the focusing of DNA with isotachophoresis in chemicalcycling PCR conditions.

FIGS. 3 a-b depict an exemplary method of on-chip chemical cycling PCR.

FIG. 4 illustrates the correlation of solvent type and concentrationwith nucleotide melting temperature using the example of 16S rRNA.

FIGS. 5 a-5 b illustrate end-point detection and real-time monitoring ofchemical cycling PCR.

FIG. 6 shows monitored DNA concentration results from an experiment.

FIG. 7 shows an example of suitable apparatus for practicing someembodiments of the invention.

DETAILED DESCRIPTION

Definitions

The term “nucleic acid” as used herein means a polymer composed ofnucleotides (“polynucleotides”), e.g., deoxyribonucleotides orribonucleotides, or compounds produced synthetically which can hybridizewith naturally occurring nucleic acids in a sequence specific manneranalogous to that of two naturally occurring nucleic acids, e.g., canparticipate in Watson-Crick base pairing interactions. The terms“nucleic acid” and “polynucleotides” are used interchangeably.

The terms “ribonucleic acid” and “RNA” as used herein mean a polymercomposed of ribonucleotides.

The terms “deoxyribonucleic acid” and “DNA” as used herein mean apolymer composed of deoxyribonucleotides.

The term “oligonucleotide” or “oligo” as used herein denotessingle-stranded nucleotide multimers up to about 400 nucleotides inlength.

Hybridization means the combination of complementary, single-strandednucleic acids into a single molecule. “Hybridizing” and “binding” areused interchangeably.

“High” denaturant concentrations mean working concentrations of morethan 10% v/v or more than 1 M.

“Low” denaturant concentrations mean working concentrations of ≦10% v/vor ≦1 M.

FIG. 1 is a conceptual representation of ccPCR. Clouds of highdenaturant concentration (one of which is labeled as 102) flow in adirection 104. A DNA template 106 has an ITP electromigration direction108 that is opposite to direction 104. As a result of this counter-flowprocess, template 106 experiences a chemical cycling that can mimic thedenaturing and then annealing effects caused by thermal cycling inclassical PCR. Locally high denaturant concentration regions meltdouble-stranded nucleic acid, while locally low denaturant regions allowfor polynucleotide annealing and extension.

During the denaturation, annealing and enzyme-aided extension process,the nucleic acid can be kept stationary in a microfluidic channel bybalancing flow velocity 104 with ITP velocity 108, while being exposedin a counter-flow stream to a series of individual clouds of movingdenaturant. This exposes the nucleic acid to alternately high and lowconcentrations of denaturant. ITP provides focusing of the nucleic acidsand protects the nucleic acids from being dispersed duringamplification. The nucleic acid can remain stationary with respect tothe laboratory frame of reference, while denaturant clouds move with thecounter-flow.

The chemical cycling process can be facilitated through spatialfluctuations in the concentration of the chemical denaturants along amicrochannel, which can be created by a flow control scheme, and resultsfrom the high electrophoretic mobility of nucleotides and the electricalneutrality of denaturants. Since denaturant clouds are electricallyneutral, they are driven forward toward, through, and then away from thenucleic acid zone. The velocity of the nucleic acid zone can be greateror less than that of the “train” of denaturant clouds. The velocity ofthe nucleic acid zones can also be zero or non-zero relative to thelaboratory frame. Preferably, the nucleic acid zone is substantiallystationary with respect to the laboratory frame. The nucleic acid zonevelocity is different than that of the denaturant clouds because of theelectric field and the differing charge state of the denaturant andnucleic acid.

The enzyme used for extension of the primer can be a heat-labilepolymerase or polymerase fragment (lacking 5′->3′ exonuclease activity),e.g. Klenow fragment of DNA polymerase I, or a thermostable polymerase,e.g. Taq DNA polymerase. Chemical denaturants can be used inconcentrations >10% to denature and open up double-strandedpolynucleotides. Chemical denaturants can be used in concentrations of0-10% to allow for and to adjust annealing and extension processes.

Polynucleotide samples can be kept stationary and confined using ITP,while moving electrokinetically through varying concentrations ofchemical denaturants. The electromigration velocity of the nucleic acidcan be balanced out by the application of a pressure-driven orelectroosmosis-driven counter flow of denaturants clouds.

Several advantages follow from this approach. PCR amplification time canbe significantly decreased. PCR system and supporting instrumentationdesign can be simplified (since no thermal cycling is required). PCRspecificity and quantitative accuracy can be increased. PCR costs can bedecreased. The tendency of the nucleic acid to be dispersed by thedenaturant flow is counter-acted by the ITP process, which ensuresstable confinement of the nucleic acids while allowing denaturantflow-through. Amplified products can simultaneously be separated by ITPwhile amplification is ongoing.

FIG. 2 shows an example of the focusing of DNA with isotachophoresis inccPCR conditions. Here LE is the leading electrolyte, TE is the trailingelectrolyte, 202 is the DNA template ITP zone, 206 is theoligonucleotide (primer) ITP zone, and 204 is the ITP zone of a nonfluorescent spacer (here benzoate) that separates the DNA from theprimers, providing PCR product localization. The use of a spacer asshown here can facilitate PCR product localization. The direction of thedenaturant counter-flow in this example is shown as 208.

FIGS. 3 a-b show an exemplary scheme of on-chip ccPCR in a cross channel302. A DNA template zone 312 is confined by ITP between a leadingelectrolyte LE and a trailing electrolyte TE. A spacer zone 314 and aprimer zone 316 are also confined by ITP between the LE and TE, asshown. The ITP velocity is shown as 310, and is opposite to thedirection of fluid flow in the channel such that the ITP zones areroughly stationary with respect to the channel.

Two consecutive denaturant clouds are shown as 306 and 308. They areseparated by PCR buffer 307, which also includes the leading electrolyteand is accordingly labeled as LE. A flow control scheme at the crossusing a denaturant reservoir 304 can be employed to provide theseperiodic clouds of denaturant.

The situation shown on FIG. 3 a is when a cloud of denaturant (i.e.,cloud 308) just enters the DNA template zone. The resulting reaction inthe ITP zones is schematically shown as double stranded DNA 320separating into its single strands 322. The situation shown on FIG. 3 bis after cloud 308 has passed through the ITP zones. The resultingreaction in the ITP zones is schematically shown as single-stranded DNA322 binding to a primer to provide primed DNA strands 324, which arethen extended to corresponding double stranded DNA 326 by a polymerase.

A series of clouds (small controlled volumes, doses or “plugs”) ofchemical denaturants can be introduced into an amplification channelwith a valve and pressure driven flow. During the denaturation,annealing and extension process, the nucleic acid is kept at a velocitydifferent than that of the “train” of denaturant clouds on themicrofluidic platform by an electric field. The nucleic acid is keptfocused in a relatively small region (relative to channel length) by anelectric field gradient that is achieved by isotachophoresis, whilebeing exposed in a counter-flow stream to clouds of moving denaturantsof alternately high and low concentrations. Locally high denaturantconcentration regions melt the nucleic acid, while locally lowdenaturant regions allow for nucleic acid annealing and extension. Theelectric field aids to focus and refocus the nucleic acid and protectsthe nucleic acid from dispersing during amplification.

The chemical cycling process can be facilitated through spatialfluctuations in the concentration of the chemical denaturants along amicrochannel, which can be created by a pressure-driven flow controlscheme, and is achieved through the high electrophoretic mobility ofnucleotides combined with the electrical neutrality of denaturants.

The DNA sample can be initially introduced into a channel sectionbetween the two electrolytes used in the isotachophoresis process. Uponapplication of the electric field, isotachophoresis focuses the sampleinto a sharp zone.

Denaturing chemical agents have the ability to reduce the meltingtemperature of DNA, i.e. the temperature at which double-stranded DNAseparates into two complementary single strands. The chemical cyclingPCR runs at a temperature at which DNA is single stranded in denaturant,but double stranded in regular buffer (with water only as solvent). Highdenaturant concentration allows denaturation, while low concentrationallows annealing and extension. The denaturants used in one particularembodiment of the invention are formamide and urea. Any other compoundsthat can act as nucleic acid denaturants can also be employed.

Non thermostable polymerases such as the Klenow fragment from E. ColiDNA polymerase I have enhanced accuracy compared to thermostablepolymerases. Also, the temperature at which their activity is optimum isclose to typical room temperature. Chemical cycling in conjunction withnon thermostable polymerase has the potential to perform DNAamplification at room temperature with high accuracy.

The denaturant injection can be controlled by pressure driven flow orelectroosmotic flow. For pressure driven flow, denaturant and otherbuffers can be connected to the inlets of the chip with capillarytubing. Flow can be generated with a hydrostatic pressure head or anautomated pressure controller. A valve allows switching off flow betweenbuffer and denaturant. Periodic switching of the valve createsdenaturant cycles within the channel. For electroosmotic injection, thechip wells can be filled with buffer and denaturant. Using a four outputpower supply, the flow can be controlled to create a succession of gatedelectroosmotic injections of denaturant, while keeping the direction ofthe electric field the same in the DNA channel throughout theexperiment.

For applications of the present approach to PCR, the PCR buffers have tobe compatible with isotachophoresis. Isotachophoresis leverages thedifference of electrophoretic mobility of ions to create electric fieldgradients. Isotachophoresis buffers are not necessarily compatible withthe PCR reaction. For PCR/ITP combinations that are untested, initialcontrol experiments to verify compatibility should be performed.

In some cases, electroosmotic flow allows better control andreproducibility of the injection and reduced dispersion compared topressure-driven counter-flow. In electroosmotic flow, the surface chargeon the channel walls originates the flow upon application of an electricfield in the channel.

In ccPCR, longer double-stranded nucleic acids can be continuouslyseparated from primer-dimers and primers during the amplificationreaction using an electrophoretic spacer (e.g., as shown on FIGS. 2 and3 a-b), while the fluorescence of the specific PCR product can bemonitored for quantitation purposes. An electrophoretic spacer is anionic species which has properties such that it ends up between thetemplate and the primers in the ITP stack of zones.

Experiment 1: On-Chip Chemical Cycling Polymerase Chain Reaction UsingFormamide and Urea as Denaturants

This assay was performed in a simple cross borosilicate glass microchip(model NS95, Caliper, Mountain View, Calif.) coated withpolyvinylpyrrolidone. Off-chip PCR buffer and denaturant reservoirs wereconnected to a low dead volume switching valve (model C2, Vici Valco,Houston, Tex.). The valve was connected to the chip with nanoports(Upchurch Scientific, Oak Harbor, Wash.) and fused silica capillaries(Upchurch Scientific, Oak Harbor, Wash.). The pressure head between theoff-chip reservoirs and the chip drives the flow. The chip was on anelectric heater maintained at 55° C. with a Peltier device and atemperature controller (Omega Engineering, Stamford, Conn.). Asourcemeter (model 2410, Keithley, Cleveland, Ohio) was used to applyhigh voltage and perform ITP.

The reaction was monitored with an inverted epifluorescent microscope(Eclipse TE300, Nikon) equipped with a cooled CCD camera (Princetoninstruments, Trenton, N.J.), and controlled with the data acquisitionsoftware V++. Images were processed with MATLAB.

A 1× PCR Mastermix (Qiagen, Valencia, Calif.) was used as the leadingelectrolyte (LE). The Mastermix contains 50 mM potassium chloride, 10 mMTris hydrochloride, 1.5 mM magnesium chloride, 2.5 U/100 μL Taq DNApolymerase, 200 μM of each dNTP. The trailing electrolyte was 25 mM TrisHEPES with 2.5 U/100 μL Taq DNA polymerase (Qiagen, Valencia, Calif.),200 μM of each dNTP (New England Biolabs, Ipswich, Mass.), and 1.5 mMmagnesium chloride. Both LE and TE contain 20 nM of forward and reverseprimers (Operon, Huntsville, Ala.), 2 μM SYTO13 intercalating dye(Molecular Probes, Eugene, Oreg.) and 0.01% Tween 20 (Sigma, St Louis,Mo.). The denaturant was 40% formamide 4M urea buffered with 1× PCRbuffer and containing 1.5 mM magnesium chloride, 2 μM SYTO13 and 20 nMof each primer. The DNA template (194 bp segment 341-534 of the 16S rRNAgene from E. Coli.) was diluted in LE solution.

A finite amount of DNA template was initially injected hydrodynamicallybetween LE and TE. Then, a voltage was applied to start ITP focusing.Once the template was focused, the voltage was adjusted so that thesample remains stationary in its channel. Then the denaturant cyclingwas started by actuating the denaturant valve. SYTO13 fluorescence wasmonitored during the denaturant cycles.

FIG. 4 illustrates the correlation of solvent type and concentrationwith nucleotide melting temperature using the example of 16S rRNA.Measured effect of formamide and urea concentration on meltingtemperature T_(m) of the 16S rRNA gene from E. Coli. T_(m) decreaseswith increasing urea and formamide concentrations. These resultsdemonstrate the effect of formamide and urea as denaturants.

FIGS. 5 a-5 b illustrate end-point detection and real-time monitoring ofccPCR. a) Isotachopherograms of PCR product zone before (left) and after40 ccPCR cycles (right). This type of end point detection allowsidentification of DNA sequences of interest. b) Real time fluorescencemonitoring of ccPCR. Initially, the PCR product fluorescence signal isbelow limit of detection.

Experiment 2: Initial PCR/ITP Control Experiments

A series of calibration experiments were performed to optimize thechemistry and flow conditions. The efficacy of Taq Polymerase wasdemonstrated for a relatively low extension temperature of 55° C. Finalconcentrations of three-step classical PCR products of a 200 bp templatefrom E. Coli by agarose gel electrophoresis were compared. The PCRtrials show very similar yields for extension temperatures of 55° C. and72° C. demonstrating high Taq activity at 55° C. for the tested template(data not shown). This result shows that annealing and extension arepossible at 55° C.

In a second set of experiments, it was verified that the ITP conditionscan result in a buffer compatible with PCR. Buffer type, bufferconcentration, and DNA concentration can have a strong effect on PCRefficiency. Ionic concentration was varied by changing the PCR bufferconcentration of a three-step classical PCR from 1× to 6× (1×PCR buffercorresponds to 50 mM KCl, 20 mM Tris-HCl) and PCR yields for eachconcentration were compared by agarose gel electrophoresis. PCR yielddecreases dramatically for buffer concentrations greater than about 3×(data not shown). Similar experiments suggest that PCRs in Tris-HEPESbuffer with concentrations ranging from 25 mM to 100 mM have similaryields to PCR in a 1×PCR buffer (data not shown).

A counter flow stream with alternately high and low denaturantconcentration was moved through ITP-confined polynucleotide samples, asin FIG. 3. The counter-flow stream can be pressure driven and/orelectroosmotic. Double-stranded DNA (dsDNA) concentration was monitoredvia SYBR Green I intercalating dye fluorescence and single-stranded DNA(ssDNA) concentration was monitored by SYBR Green II fluorescence. Afused silica microchip with silanized channels was employed, and Tween20 was added to all solutions to minimize protein adsorption.

FIG. 6 is a plot of DNA concentration monitored via SYBR Green Ifluorescence plotted versus time. Under conditions of balancedelectromigration and bulk flow velocity, the DNA plug oscillates withinabout a 1 mm region. Fluorescent intensity decreases dramatically duringdenaturing before increasing as DNA amplifies via annealing andextension.

FIG. 7 shows an example of a device 702 suitable for practicing theabove-described ITP/PCR process. A micro-fluidic chip 708 having crossedchannels 710 and 712 sits on a plate 704 maintained at a constanttemperature of 55° C. at which denaturant activity is optimum. Thetemperature of plate 704 can be maintained by a Peltier device 706attached to a heat sink (not shown). Electric field can be applied fromHV well 724 to GND well 726 to effect ITP focusing of DNA. PCR bufferflows continuously into chip PCR well 728 via hydrostatic pressure.Denaturant flows into well 722 under control by a valve (not shown) thatis actuated in pulses causing small denaturant injection clouds to flowtoward the HV well. Voltage is preferably controlled to hold the DNAband approximately stationary via ITP dynamics. The chemicalconcentration cycling amplification process can monitored in real timeby measuring intensity of fluorescence 732 at the end of eachamplification cycle with a monitor 730. Real time fluorescencemonitoring can be performed with an epifluorescent microscope and acomputer controlled CCD camera. ccPCR product quantity can be determinedby intercalating dye fluorescent or by sequence specific fluorescentprobe such as molecular beacons.

The preceding description has been by way of example as opposed tolimitation, and various modifications of the given examples also rely onthe above-described principles. In particular, the preceding examplesrelate to the various chemical processes associated with the polymerasechain reaction. However, the use of ITP to control chemical reactionscan be applied to any chemical reaction, not just PCR. The precedingexamples also relate to the use of micro-fluidic devices. However,standard capillaries and interconnects can also be employed.

The invention claimed is:
 1. A method of performing a chemical reaction,the method comprising: providing a first reactant confined byisotachophoresis (ITP) to a first ITP zone in a liquid flow channel;providing a second reactant which is not confined to said first ITP zoneby said isotachophoresis and which flows through said first ITP zone;allowing said first and second reactants to react in said first ITP zoneto provide at least a first product.
 2. The method of claim 1, whereinsaid first ITP zone is substantially stationary in said liquid flowchannel.
 3. The method of claim 1, further comprising monitoring aconcentration of at least one of said reactants or said first product.4. The method of claim 1, further comprising providing a catalyst orenzyme in said first ITP zone.
 5. The method of claim 1, wherein saidchemical reaction comprises a polymerase chain reaction, wherein saidfirst reactant comprises nucleic acid, and wherein said second reactantcomprises nucleic acid denaturant.